The present invention relates generally the field of PEGylated transforming growth factor alpha (TGF-xcex1) polypeptides, related polypeptides and fragments thereof and the use of such compounds for a variety of pharmaceutical purposes, including for stimulating stem cells proliferation, migration and differentiation.
Stem cells and tissue precursor cells play important roles in the development, regeneration and repair of organisms and particularly tissue and organs. Stimulation of tissue regeneration and repair can provide needed benefit to organisms suffering from injury, disorders or diseases which impair physiological functions increasing mortality and morbidity. For example, there are several disease treatments that could significantly benefit by having cells regenerate after injury or lesion formation. For example, in some instances, a particular treatment for a disease often detrimentally affects the subject being treated. One such example, is the administration of chemotherapeutic agents to subjects, which results in destruction of healthy cells, for example, cells of the gastrointestinal tract. Such chemotherapeutic agents include carmustine (BCNU), chlorambucil (Leukeran), cisplatin (Platinol), Cytarabine, doxorubicin (Adriamycin), fluorouracil (5-FU), methoxetrate (Mexate), taxol, CPT111, etoposide, and plicamycin (Mithracin) which are known for their direct stomatotoxic potential (Sonis, 1993, xe2x80x9cOral Complications in Cancer Therapy,xe2x80x9d In: Principles and Practice of Oncology, pp. 2385-2394, DeVitta et al., Eds., J. B. Lippincott, Philadelphia) and hence incidence of mucositis.
Oral mucositis is an example of a disorder resulting from the cytotoxic effects of chemotherapy and/or radiotherapy on the rapidly dividing epithelial cells of the oropharyngeal mucosa, and is exacerbated by infection with both endogenous oral flora and opportunistic bacterial and fungal pathogens. Complications related to oral mucositis vary in the different patient populations affected, but typically include pain, poor oral intake with consequent dehydration and weight loss, and systemic infection with organisms originating in the oral cavity. The pain associated with oral mucositis may be severe requiring narcotic analgesics, and the difficulty in eating can result in patients receiving total parenteral nutrition.
Accordingly, adult cell renewal via the differentiation of immature cells can help to alleviate the problems associated with tissue and cellular damage. For example, stimulating renewal of blood and lymphoid cell types by stimulating developmentally immature precursors (hematopoietic stem and progenitor cells) can assist in treating disorders associated with blood or lymphatic cell depletion.
While the hematopoietic system is the best understood self-renewing adult cellular system, it is believed that most, perhaps all, adult organs harbor precursor cells that under the right circumstances, can be triggered to replenish the adult tissue. For example, the pluripotentiality of neural crest cells and tissues of the adult gut contain immature precursors which replenish the differentiated tissue. The liver has the capacity to regenerate because it contains hepatic immature precursors. In addition, various epithelial cells, including cells of the skin renew due to proliferation and differentiation of stem cells. Through the mesengenic process, most mesodermal derivatives are continuously replenished by the differentiation of precursors. Such repair recapitulates the embryonic lineages and entails differentiation paths which involve pluripotent progenitor cells.
Mesenchymal progenitor cells are pluripotent cells that respond to specific signals and adopt specific lineages. For example, in response to bone morphogenic factors, mesenchymal progenitor cells adopt a bone forming lineage. For example, in response to injury, mesodermal progenitor cells can migrate to the appropriate site, multiply and react to local differentiation factors, consequently being induced down a distinct differentiation path. It has been suggested that the reason that limited tissue repair is observed in adults is because there are too few progenitor cells which can adopt specific differentiation lineages. It is clear that if such progenitor cells could be expanded tissue repair could be occur much more efficiently. In addition, an expanded pool of stem and progenitor cells, as well as non-terminally differentiated cells supplying a desired differentiation phenotype, would be of great value in transplantation and gene therapy as well as a myriad of therapeutic regimens.
The invention provides methods and compositions useful for expanding precursor cell and stem cells, including adult stem cell populations in vitro, in vivo, or ex vivo or a combination thereof. The methods and compositions are useful in treating a number of diseases, disorders or injuries by inducing the expansion, migration and differentiation of precursor cells, stem cells or adult stem cells.
In one embodiment, the invention provides a method for protection of a tissue or an organ from damage by a cytotoxic agent or other injury causing agent or activity. For example, cytotoxic chemotherapy drugs cause gastrointestinal toxicity manifested by nausea, vomiting and diarrhea. In another example, HIV infection, AIDS and current therapies cause diarrhea in approximately 80% of patients. The method includes administering an effective amount of a TGF-xcex1 polypeptide (SEQ ID NO:1), a TGF-xcex1 related polypeptide, a TGF-xcex157 polypeptide (SEQ ID NO:3), a functional fragment thereof or a mimetic thereof to the tissue or organ prior to, simultaneously with or subsequent to contacting the tissue or organ with the cytotoxic agent or the injury causing agent or activity. Tissues include gastrointestinal tissue, urogenital tissue, musculoskeletal tissue, nerve tissue, or cardiovascular tissue, for example.
In another embodiment, the invention provides a method for treating, regenerating or repairing a tissue of a subject in vivo. The method includes contacting a tissue with a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a fragment or a mimetic thereof prior to, contemporaneously with, or subsequent to a tissue injury in an amount effective to induce stem cell or precursor cell proliferation, migration, or differentiation at the site of injury thereby treating, regenerating or repairing the tissue. Contacting may be as a continuous infusion or by a bolus or single administration, for example.
In yet another embodiment, the invention provides a method for treating or preventing mucositis of the gastrointestinal tract in a subject. The method includes administering a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a TGF-xcex157 polypeptide, a fragment thereof, or a mimetic thereof in an amount effective to treat, inhibit or prevent gastrointestinal mucositis in the subject.
In another embodiment, the invention provides a method for expansion of a precursor cell, stem cell, or adult stem cell by recombinantly expressing within the cell an amount of a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a functional fragment thereof, or a mimetic thereof effective to induce proliferation of the cell; and culturing the cells under conditions such that the cell proliferates.
In another embodiment, the invention provides a method for expansion of a hematopoietic precursor cell, an epithelial precursor cell, or a liver precursor cell, for example. The method includes recombinantly expressing within the cell an amount of a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a functional fragment thereof, or a mimetic thereof effective to induce proliferation of the cells; and culturing the cell under conditions such that the cell proliferates. Preferably, the precursor cell is a human cell.
In another embodiment, the invention provides a method for promoting mammalian neuronal cell growth by contacting a mammalian neuron in vitro with a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a functional fragment thereof, or a mimetic thereof effective to induce proliferation of the cells; and culturing the cells under conditions such that the cell proliferates.
In yet another embodiment, the invention provides a method for expanding stem cells ex vivo. The method includes culturing stem cells from a subject and contacting the stem cell culture with a TGF-xcex1 polypeptide, a TGF-xcex1 related polypeptide, a functional fragment thereof, or a mimetic thereof in an amount necessary to augment stem cell growth.
In another embodiment, the present invention provides a method for treating Type I or Type II diabetes by expanding a subject""s population of insulin-producing cells. The method includes administering an effective amount of a TGF-xcex1 polypeptide (SEQ ID NO:1), a TGF-xcex1 related polypeptide, a TGF-xcex157 polypeptide (SEQ ID NO:3), a fragment thereof, or a mimetic thereof in an amount effective to expand the population of insulin-producing cells.
Also provided is a method for treating AIDS or HIV infection by increasing a subject""s population of CD4+ T cells. The method includes administering an effective amount of a TGF-xcex1 polypeptide (SEQ ID NO:1), a TGF-xcex1 related polypeptide, a TGF-xcex157 polypeptide (SEQ ID NO:3), a fragment thereof, or a mimetic thereof.
Also provided are TGFxcex1 mimetics that are PEGylated TGF-xcex1, related polypeptides and fragments thereof for use in the pharmaceutical methods recited herein. For the purposes of the present invention, the TGF-xcex1 may be human (SEQ ID NO:1) or of other mammalian origin. For the purposes of the present invention, TGF-xcex1 related polypeptides include those that share homology or the three loop, cysteine-bonded structure with TGF-xcex1 as described herein. In a preferred embodiment of this aspect of the present invention, there is provided a PEGylated human TGFxcex1 polypeptide of the formula:
xe2x80x83(R)n-Txe2x80x2xe2x80x83xe2x80x83(VIII)
wherein R is a PEG group, n is 1 or 2, Txe2x80x2 is selected from one of the following:
a polypeptide of SEQ ID NO:1;
a polypeptide extending from amino acid #2 (Val) to amino acid #50 (Ala) of SEQ ID NO:1;
a polypeptide extending from amino acid #3 (Ser) to amino acid #50 (Ala) of SEQ ID NO:1;
a polypeptide extending from amino acid #4 (His) to amino acid #50 (Ala) of SEQ ID NO:1;
a polypeptide extending from amino acid #5 (Phe) to amino acid #50 (Ala) of SEQ ID NO:1; or
a polypeptide extending from amino acid #6 (Asn) to amino acid #50 (Ala) of SEQ ID NO:1;
and wherein R is attached to Txe2x80x2 at the amino-terminus, at amino acid #29 (Lys) of SEQ ID NO:1 or both.
Preferred PEG groups are also described herein and form part of this aspect of the present invention.